SP3: Thorsten Pfirrmann - The role of protein glycation on proteostasis.

Background and previous workAgeing cells accumulate protein aggregates due to a decline of 20S/26S proteasomedependent protein degradation. Proteolytic insufficiencies are also involved in age-related diseases like Parkinson or Alzheimer disease and cataract. The reason for this phenomenon is unknown, but several observations suggest that posttranslational protein modifications like glycation play a role. For example, an age-related increase in the content of glycated proteins and protein aggregates showed an inverse relationship to lifespan. However, it is not understood why glycated, misfolded proteins cannot be degraded via the 20S/26S proteasome and accumulate over time. A better understanding Research Programme 13 of the molecular mechanisms of age-related protein degradation deficiencies is necessaryto develop novel therapies for patient treatment.Specific aimsOur goal is to experimentally address several questions: (1) Are glycated proteins targeted by the 20S/26S proteasome for degradation and (2) is there a general stress response to glycation, e.g. upregulation of ubiquitin proteasome system components (e.g. ubiquitin,Ubc4/5, p97/Cdc48, ubiquitin ligases (E3s))? (3) Does protein glycation directly interfere with polyubiquitination at lysine residues?Working programmeIn a first step we plan to address these questions with different proteasomal substrates, e.g. fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase (PEPCK), ornithinedecarboxylase (ODC; ubiquitin-independent) and artificial lysine-less proteasomal substrates in Saccharomyces cerevisiae. Later, custom-made in vitro green fluorescent protein (GFP)-reporters will be used to measure ubiquitin-proteasome-system activity. Glycation will be introduced chemically by the addition of methylglyoxal in vivo and in vitro. Different sugars (galactose, xylose) and derivatives (6-dehydroxy-glucose, glucuronic acid) will be used to induce glycation in yeast. Glucose metabolising enzymes like FBPase and PEPCK are known targets of endogenous carbonylation in ageing cells. Therefore, we plan to study the ubiquitination/carboxyethyl-lysine status and activity of both enzymes in replicative and chronological aged yeast cells. To find ubiquitin-proteasome-system components essential for the degradation of glycated proteins, we plan to use conditional temperature sensitive yeast cells to deplete essential 20S/26S proteasome subunits and Cdc48, or deletion strains of different ubiquitin conjugating enzymes (e.g. Ubc4/5). Substrate modification and protein half-life will be detected by co-immunoprecipitation, subsequent Western blotting and staining with antiubiquitin and e.g. anti-CML antibodies or by mass spectrometry. To look for putative stress response genes, it is planned to perform RNA deep-sequencing of treated versus untreated cells. Collaborations are planned with SP1, SP2, SP5 and SP10 with respect to analyses of glycation, O-GlcNAc and sialic acid, and with SP6 and SP13 concerning stress response mechanisms and metabolic characterisation, respectively.